Gene transcription underlies development, oncogenesis, and the constant reshaping of the cell in response to a variety of metabolic and environmental cues: many morphogens and oncoproteins are transcriptional activators, and transcription is the endpoint of a number of signal transduction pathways. The basal transcription machinery comprises RNA polymerase II (the enzyme responsible for synthesis of all messenger RNA in eukaryotes) and five accessory proteins, known as general transcription factors. RNA polymerase II and the general transcription factors assemble at a promoter to form a preinitiation complex, which comprises over 30 different polypeptides and has a molecular mass of almost 2 MDa. The size and complexity of the preinitiation complex have prevented conventional techniques such as X-ray crystallography and NMR spectroscopy from providing structural information about it. The overall goal of this project is to employ macromolecular electron microscopy and image analysis (techniques that are ideally suited for the structural characterization of large, scarce macromolecular assemblies) to reveal the structure of the yeast and human preinitiation complexes under physiologically-relevant conditions. A step-wise approach will be applied: structures of progressively larger complexes formed by RNA polymerase and the general transcription factors will be determined, following the order of assembly of the preinitiation complex in vivo. The structural changes that RNA polymerase undergoes as the general transcription factors assemble, and the topology of the entire preinitiation complex, will reveal the mechanism of transcription initiation and promoter escape. The preinitiation complex is the target of Mediator, a multiprotein complex that is essential for response of the basal transcription machinery to regulatory signals from activators and repressors. We will complete our work by determining the structure of the Mediator/preinitiation complex assemblies in the presence of activators and repressors. The structures will indicate the way in which regulatory information is transduced to the preinitiation complex, and reveal the structural underpinnings of the transcription regulation mechanism in eukaryotic cells.